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Protein Interactions

The scaffold protein mAKAP is an example of an AKAP that is well characterized. Moreover, mAKAP provides an example of how AKAPs function to link multiple signaling pathways and to control local cAMP levels. mAKAP is a 230-kDa protein present at the nuclear envelope in both cardiac and skeletal myocytes (Kapiloff et al. 1999 Michel et al. 2005). The alternatively spliced-form mAKAPa is present in neurons. mAKAP complexes can contain PKA, the phosphodiesterase PDE4D3, the calcium-activated...

Lamin proteins are type-V IF proteins that, unlike other family members, assemble to branched filaments. Besides the cage-like framework at the inner nuclear membrane, lamins are also found throughout the nucleus fusions of lamins A and B with GFP reveal a homogenous nucleoplasmic veil in addition to the intensely fluorescing nuclear lamina. Mobility measurements show that within the veil lam-ins are more mobile and somewhat less resistant to the conventional extraction steps. Jackson (2005)...

AKAP-Lbc is the second AKAP that is abundant in cardiac tissue and has been implicated in hypertrophic signaling pathways involving Rho and PKD (Fig. 4). AKAP-Lbc is the full-length form of the PKA-binding fragment Ht31 that was first isolated from human thyroid and has been widely used as a prototype to study the structural basis of PKA-AKAP interactions (and the physiologic importance of AKAP-mediated PKA anchoring in physiologic responses). AKAP-Lbc is a large 320-kDa modular protein with an...

Listeria monocytogenes infections are a common cause of food poisoning, and an effective means to block their infectivity is highly desirable. These pathogens contain a protein called ActA that contains four EVH1 FPPPP consensus motifs and that effectively hijacks the EVH1-containing proteins Ena and VASP (Chakraborty et al. 1995). EVH1-mediated actin binding allows the bacterium to propel itself along the cell's actin cytoskeleton, leaving behind a so-called actin-rich comet tail and thus...

ErbB-4 is the newest identified member of the epidermal growth-factor-receptor family and seems to be a good tumor marker for certain types of malignancies, such as prostate, lung or breast cancer (Starr et al. 2006 Ben et al. 2007 Junttila et al. 2005). The C-terminal fragment (CTF) of ErbB-4 is cleaved by y-secretase and shuttles to the nucleus of stimulated cells (Ni et al. 2001). The two WW-domain-containing proteins WWOX and YAP (Yes kinase associated protein) were shown to compete for...

The first unimolecular FRET probes for cAMP exploited Epac as the sensor for the cyclic nucleotide (DiPilato et al. 2004 Ponsioen et al. 2004). One of these indicators was generated by fusing the amino terminus of the Epac 1 protein to CFP and the C-terminus to YFP (Ponsioen et al. 2004). Such sensors localize to the cytosol and to membranes, in particular to the nuclear envelope and to perinuclear compartments (Ponsioen et al. 2004). To generate a cytosolic variant, the DEP domain (amino acids...

The GYF domain has so far not yet been unambiguously assigned to certain disease states. Nevertheless, overexpression of the GYF-containing protein CD2BP2 was observed in chronic fatigue syndrome (Kaushik et al. 2005), and the same protein was found to be overexpressed in estradiol-, 4-hydroxytamoxifen- and acolbifene-treated T47D breast cancer cells (Al Dhaheri et al. 2006). Independent of its possible role in these disease-related processes, GYF domain inhibition would be a valuable tool for...

The rapid calcium influx of phase 2 of the cardiac myocyte action potential incites CICR from intracellularly sequestered, sarcoplasmic reticular calcium stores via (predominantly) ryanodine receptor (RyR)-mediated release. RyRs are the largest ion channels characterized to date homotetramers consisting of four monomers that are each 560 kDa and have large cytoplasmic domains (Wehrens and Marks, 2005 Santonastasi and Wehrens, 2007). These cytoplasmic domains also serve as the junctions at which...

Peptides mimicking binding domains disrupt protein-protein interactions with high selectivity by competitively binding to one of the interacting partners. Such peptides have been widely utilized to gain insight into the function of protein-protein interactions (Shin et al. 2005). The generation of disruptor peptides requires mapping of the interacting domains. Recent studies took advantage of a combination of peptide spot technology (Kramer and Schneider-Mergener 1998 Frank 2002) and overlay...

AKAP350 targets PKA and a variety of interacting proteins to the Golgi apparatus in epithelial cells. Larocca et al. utilized RNA directed against AKAP350, which reduced the expression level to 20 in HeLa cells and disperses the Golgi apparatus, indicating that the AKAP is important for the maintenance of the Golgi. The human AKAP79 and its rat ortholog AKAP150 bind PKA, PKC and calcineurin. Knockdown of AKAP79 in HEK293-B2 cells, stably expressing the P2-adrenoceptor, revealed that...

Transcription of immune regulatory genes involves the coordinated action of several transcription factors, including NFAT, NF-kB, API and members of the forkhead box family (Coffer and Burgering 2004 Kuo and Leiden 1999). It is therefore relevant to examine how receptor proximal events are linked to these mediators and how specificity in the regulation of the different transcription factors is maintained. There is accumulating evidence demonstrating that fine-tuning of signals at this level in...

The N-terminal region of the long-form human isoform, PDE4A4, has been shown to be important in directing localization of this protein and also to play a role in determining the inherent sensitivity of the enzyme to the archetypical PDE4 inhibitor, rolipram (Huston et al. 1996). PDE4A4 is distributed within both the particulate and cytosolic fractions of cultured COS7 cells. This localization was shown to have a profound effect on the inhibition of the enzyme by rolipram, with the particulate...

A central challenge of forward chemical genetic screens is the identification of protein targets of small-molecule inhibitors (Stockwell 2000 Burdine and Kodadek 2004 Tochtrop and King 2004). Although a number of conceptually distinct approaches have been used successfully for inhibitor target identification (Burdine and Kodadek 2004 Tochtrop and King 2004), no single method has proven universally applicable. To identify the target of wiskostatin, we therefore began with a candidate-testing...

The potential to interfere with PPIs in multi-protein signaling complexes is of great importance both for the understanding of cell physiology and for the development of novel disease treatments. Modular proteins involved in signal transduction utilize highly conserved non-catalytic adaptor domains that mediate PPIs during the formation of multi-protein signaling complexes. In general, these adaptor domains must fulfill certain requirements to exert their binding function. Firstly, the...

A small group of receptors implicated in immune cell regulation are members of the signaling lymphocytic activation molecule (SLAM) family, which belongs to the CD2 subfamily of Ig receptors. These include SLAM (CD150), 2B4, CD84, NTBA (NK-,T- and B-cell antigen), Ly9 (CD229) and CD2-like receptor activating cytotoxic cells or CRACC (CD319) (Ma et al. 2007 Veillette 2006). They have Ig-like domains in their extracellular regions and are expressed in different subsets of lymphocytes. Except for...

In addition to the intramolecular interactions that regulate Pakl activity, there are a number of other proteins that interact with and modulate Pakl activity. Interestingly, many of these proteins bind within the Pakl autoregulatory region. For example, human Pakl-interacting protein (hPIPl) is a WD repeat containing protein that was shown to inhibit Pakl kinase activity as well as the signaling of Pakl effectors JNK and NFkB in human cells. hPIPl binds within the autoregulatory region of...

Fig. 1 Peptide substitution array of the RII-binding domain of AKAP18S. Shown is a peptide array of spot-synthesized 25-amino-acid-long peptides, comprising the RII-binding domain of AKAP18S (positions 296-320 vertical) in which every amino acid is replaced by the amino acids indicated at the top of the membrane. Peptides were spot-synthesised on cellulose membranes and probed for binding to 32P-labelled RII subunits of PKA (RII-overlay assay). Binding of RII-subunits was detected by...

A major challenge in the actin field is the difficulty of determining the contribution of different classes of actin filament nucleators to the generation of new actin filaments because of the presence of other abundant actin filament nucleating activities. A small-molecule inhibitor that is specific for the formin family would therefore be useful in discriminating the relative contribution of the formins in the context of the three classes of actin nucleators (Arp2 3 complex, Spire, and...

The mammalian PDE4 family comprises around 20 different isoforms encoded by four genes, PDE4A, PDE4B, PDE4C and PDE4D (Conti et al. 2003 Houslay and Adams 2003 Houslay et al. 2005) (Fig. 2). Each gene is large and complex, spanning around 50 kb and having around 20 exons, which gives rise to multiple isoforms by alternative mRNA splicing (Monaco et al. 1994 Bolger et al. 1997). Each isoform has a modular structure consisting of an isoform-specific N-terminal region, all or part of Fig. 2 The...

Transmembrane adaptor proteins (TRAPs) are a group of integral membrane proteins that recruit and assemble signaling molecules proximal to the antigen receptors (Horejsi et al. 2004). These molecules are characterized by short extracellular domains followed by a transmembrane domain and long cytoplasmatic tails containing mainly tyrosine-based interaction motifs. The phosphorylation status of these motifs changes after immunoreceptor ligation and Zap-70 activation, thereby regulating their...

Much of what has been discussed to this point applies only to the DAG-responsive PKC isoforms, which include the conventional (a, pi, piI, y) and novel (5, e, 8, n) subgroups. Two atypical PKC isoforms (X and Z) live in a world of their own, including a unique set of scaffolds and a distinct relationship with lipids (Suzuki et al. 2003 Moscat and Diaz-Meco 2000 Hirai and Chida 2003). The atypical PKCs are classified within the PKC family on the basis of modest sequence homol-ogy in the kinase...

Proteins, lipids, and other biomolecules are transferred among different cellular compartments by transport vesicles, which carry hydrophilic cargoes in the aqueous lumen or hydrophobic ones in the lipid membrane. Crucially, each vesicle must pinch off from the membrane of its parent compartment and recognize and fuse with that of its target compartment in a highly specific and tightly regulated way, such as to release its contents at the right place at the right time (Bennett 1995). In...

A first strategy for PRM PRD inhibition was based on the introduction of non-natural amino acids into the known wild-type peptides (Nguyen et al. 1998, 2000). They demonstrated that the second proline residue of the PxxP motif recognized by SH3 domains could be replaced with -alkylated glycine derivatives to create ligands exhibiting enhanced affinities. Their strategy for inhibitor design can be outlined as follows maintain the required hybrid Ca- and N-substituted scaffold, but vary...

Since wiskostatin inhibits the activity of full-length N-WASP, but not that of the isolated VCA domain, this suggested that wiskostatin might perturb the conforma-tional changes in N-WASP that accompany activation. How it might do so was of particular interest because N-WASP is a scaffolding protein not previously known to bind a small molecule. Available three-dimensional structural data for N-WASP that could facilitate prediction of binding sites for wiskostatin are limited to crystal...

UEV (ubiquitin E2 variant) domains are homologs of ubiquitin E2 ligases that lack the active-site cysteine, but retain the capability of ubiquitin binding, which is often of functional importance. Human UEV1a as well as its yeast homolog Mms2, for example, are involved in polyubiquitin chain assembly (Hofmann and Pickart 1999 Deng et al. 2000 VanDemark et al. 2001). Interaction with proline-rich motifs, however, has only been described for the UEV domain of human Tsg101 (tumor-susceptibility...

In order to study the function of PKA anchoring in cell and animal models, disruptor peptides have been genetically encoded for expression. Generally, this approach overcomes limitations of peptides, such as their short half-life if applied to animals parenterally. In addition, this technique allows for introduction of peptides into cells and possibly animals that are not or are only limitedly accessible to chemical synthesis. Furthermore, encoding peptides genetically permits labelling with...

Several AKAPs have been implicated in the coordination of cardiac hypertrophy. The muscle-specific AKAP (mAKAP, the 255-kDa muscle A-kinase anchoring protein) is a multivalent scaffold that binds PDE4D3 (and other signaling proteins, see below), is tethered to the nuclear envelope (through a protein-protein interaction with the integral nuclear membrane protein nesprin-1a), and plays a crucial role to synchronize PKA and PDE4D3 activity in cardiomyocytes (Fig. 3 Pare et al. 2005 Dodge-Kafka et...

Several interactors of the Shank PDZ domains have been suggested to play a role in morphogenic signaling within spines. Most notable, the guanine nucleotide exchange factor PPIX is attached to Shank via this type of interaction, involving the C-terminal PDZ ligand motif of PIX and an additional leucine zipper, also located in the C-terminal region of PPIX (Park et al. 2003). PPIX is an exchange factor for rho GTPases, in particular for cdc42 and rac. Both GTPases have been implicated in the...

DNA-damaging agents belong to the most widely used anti-cancer drugs. Depending on the extent of damage, the cells are either arrested in the cell cycle to allow DNA repair or the cells undergo apoptosis. The tumor suppressor p53 is a primary sensor of genotoxic stress and induces cell-cycle arrest or apoptosis (Vousden 2002). A major obstacle to an efficient anti-cancer therapy is the activation of NF-KB-mediated pro-survival signaling by DNA-damaging agents. There is a complex crosstalk...

Although knockout and RNA strategies have revealed crucial functions of AKAPs in elementary processes whose dysregulation causes disease, it is not clear to which degree the loss of the AKAP function of a protein, i.e. its ability to interact with R subunits of PKA, contributes to the phenotypes because gene knockout deletes all functions of a protein. Knockout of AKAP149 (also termed AKAP1) decreases fertility in female null mutant mice, but not in heterozygotes (Newhall et al. 2006). Oocytes...

Selective targeting of arrestin interactions with individual non-receptor partners has a tremendous therapeutic potential. Arrestin functions affect a huge variety of signaling mechanisms (Lefkowitz and Shenoy 2005 Gurevich and Gurevich 2006a), some of which may underlie the pathology of multiple diseases. For example, increases in arrestin expression in the brain of MPTP-treated monkeys with Parkinsonian symptoms (Bezard et al. 2005) and human patients with Parkinson's disease-dementia...

Obviously, S MARs map to non-random locations in the genome. They occur at the flanks of transcribed regions, in 5'-introns and telomeres, and also at gene breakpoint cluster regions (review Bode et al. 1995). S MARs are association points for common nuclear structural proteins (review Bode et al. 2000b and below) and proved to be required for authentic and efficient chromosomal replication and transcription, for recombination and chromosome condensation. These are the levels of their firmly...

Historically, most AKAPs were initially identified in screens for PKA-binding proteins. Thus, these AKAPs tend to be better characterized. The function and or identity of many other putative AKAPs, however, remains obscure. For cellular processes where it has been suspected that PKA anchoring may be involved, techniques to globally disrupt PKA anchoring in a cell have proven quite useful. A peptide that mimics the Rll-binding domain of AKAP-Lbc (termed Ht31 peptide) competes with AKAP PKA...

The EVH1 domain adopts a structure similar to pleckstrin homology (PH) domains. However, EVH1 domains do not bind to phospholipids. Instead, they mediate specific protein-protein interactions with proline-rich short peptide motifs. This interaction is important for the recruitment of EVH1 domain-harbouring proteins to specific subcellular locations and to membrane receptor complexes. According to their ligand specificity, they can be divided into four classes. Class-I EVH1 domains bind to a...

Coiled-coil interactions are essential not only for vesicular trafficking during intra-cellular protein and lipid transport and intercellular communication using neuro-transmitters (see Sect. 2.2), but they also constitute an indispensable part of the fusion machinery of enveloped viruses. Viruses have to overcome the membrane of the host cell to deliver their genetic material into the cytosol and nucleus so that a new infectious cycle can be established. Viral entry into host cells proceeds...

Mattern et al. (1997 and references therein) have identified the most abundant proteins that are exclusively present in the internal nuclear network. In line with earlier reports (Nakayasu and Berezney 1991), many of these belong to the group of heterogeneous nuclear ribonucleoproteins (hnRNPs), the sites of nascent transcripts and RNA maturation. These findings support a model in which major matrix protein constituents are involved in RNA metabolism, packaging and transport. The most abundant...

In the nucleus, the formation of the Tcf-P-catenin complex is a prerequisite for target gene activation. Targeted interference with this complex formation in colon cancer cells has been shown to effectively abrogate activation of target genes and to inhibit their growth in vitro (Tetsu and McCormick 1999 van de Wetering et al. 2002). Accordingly, small compounds that can specifically disrupt the Tcf-P-catenin complex in vivo hold immense potential for the treatment of Wnt-driven tumors. Earlier...

The traditional model of PAR activation focuses on the role of the heterotrimeric Gs protein to activate the cAMP pathway. PAR activation also leads to the liberation of GPy dimers that contribute to the recruitment of GRKs to the membrane, leading to phosphorylation of agonist-activated PARs (Fig. 1b). P-arrestin then binds GRK-phosphorylated PARs and acts both to sterically interdict further G protein signaling and to promote PAR desensitization. However, P-arrestins also act as scaffolds for...

PDE4 enzymes have been the subject of extensive research because of the therapeutic potential of PDE4 inhibitors to treat a number of major disease areas. However, PDE4 inhibitors have been slow to fulfill this therapeutic potential because their use is associated with dose-limiting side effects such as nausea and vomiting. Despite concerted efforts to refine PDE4 inhibitors in order to improve efficacy and minimize side effects, current inhibitors are still largely non-selective. The universal...

Due to the pleiotropic effects of cAMP signaling, it is no surprise that current therapies for some human diseases include the use of drugs that affect cAMP levels. For example, P-adrenergic agonists are a mainstay of asthma therapy due to cAMP-mediated relaxation of bronchial smooth muscle. In a complementary fashion, caffeine and theophylline have been used to inhibit phosphodiesterases. The problem with current cAMP-directed therapies is that many such drugs have unacceptable side effects....

Current methods used to eliminate Pak1 function include classical genetic knockout Hofmann et al. 2004 as well as transfection of interfering reagents, such as siRNAs Tang et al. 2005 , constructs encoding the IS and KI sequence elements Beeser and Chernoff 2005 , or catalytically inactive, full-length versions of the kinase Adam et al. 2000 . These approaches, while useful in abrogating Pak1 activity, are limited in their degree of control of Pak1 activity, as well as by a lack of control over...

A number of studies have begun to unravel the mechanism of mDial autoinhibition. Early cellular studies utilizing truncated mDial constructs demonstrated the involvement of both N-terminal and C-terminal domains in the regulation of mDia-mediated actin filament assembly Watanabe et al. 1997 Tominaga et al. 2000 Alberts 2001 . Subsequently, these regulatory activities were discovered to be the result of an interaction between a domain in the amino terminus of mDial termed the diaphanous...

A common property of all PRMs is that they preferentially form a left-handed poly-proline type-II PPII helix with an overall shape resembling a triangular prism Kay et al. 2000 . This structural element has a helical pitch of 9.3 , three residues per turn, and F and Y angles centered around -75 and 145 , respectively Bochicchio and Tamburro 2002 Cubellis et al. 2005 . The PPII helix is characterized by a complete lack of main-chain hydrogen bonding patterns that are commonly used to identify...

Protein-protein interactions preside over the physiological and pathological processes in living organisms. Therefore, the ability to intervene in a controlled manner in the protein interaction network, altering its wiring by specifically favoring certain interactions and blocking others, is crucial for molding the cell response into a desired phenotype. Examples of the efficacy of such strategy is witnessed by the modus operandi of some known pathogens, such as the Epstein-Barr virus EBV...